DESCRIPTION: The PI has proposed a staging system for fiber cell development that recognizes three distinct phases; the elongation phase, the maturation phase and the organelle degradation phase. Hereditary and congenital cataracts are often characterized by the failure of fiber cells to complete this sequence. This project focuses on the cell and molecular mechanisms that regulate progression through the sequence. He will examine the fate of fiber DNA during differentiation. The integrity of the chromatin will be measured by labeling techniques and the ability of the nucleus to support RNA transcription. This will show whether DNA degradation occurs suddenly at the border of the organelle-free zone. The investigator will examine whether fiber cell denucleation represents 'physiological apoptosis' by evaluating the condition of the lens fiber chromatin, determining whether specific apoptotic substrates are cleaved, and testing whether organelle loss requires protein synthesis. The maturation phase is characterized by increased staining for vinculin, actin and paxillin and decreased staining for N-cadherin and band 4.1 protein. He will use these markers to determine whether detachment from the posterior capsule is necessary and/or sufficient to trigger entry into the maturation or organelle degradation phases. He will examine the relationship between organelle loss and ionic homeostasis, using ratio imaging to measure Ca2+ and pH in differentiating lens fibers and ionophores to manipulate the cytosolic concentrations of these ions. He will investigate the involvement of calpains and/or ubiquitin-mediated proteolysis in organelle loss by evaluating the condition of endogenous substrates and using function-blocking antibodies to specifically inhibit the proteolytic pathways.